This invention relates to nucleic acids and, more particularly, to novel nucleic acid crosslinking agents and the use of such crosslinking agents in the affinity inactivation of nucleic acids.
All living organisms contain nucleic acids in the form of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). These nucleic acids, which consist of polynucleotide chains of varying nucleotide sequences, contain the information which directs all forms of life on our planet. It is generally accepted that during information transfer processes within cells, i.e., during replication (DNA synthesis), transcription (RNA synthesis), and translation (protein synthesis), nucleic acids exist in two different transient states, i.e., in a single-stranded configuration and in a double-helical configuration wherein two chains or strands of complementary nucleotide sequence are based-paired to each other. During the information transfer processes, the base-paired chains readily dissociate from each other into the single-stranded configuration, and each chain then serves as a template for the synthesis of two complementary chains, each newly synthesized chain then becoming base-paired to its complementary template chain.
Since the information transfer processes rely upon the ability of the base-paired chains to readily dissociate into the single-stranded configuration, the formation of interstrand crosslinks between the base-paired chains which prevent such dissociation from occurring renders the nucleic acids incapable of being replicated, transcribed or translated, and hence biologically dead. A number of bifunctional chemical compounds are known which are capable of crosslinking certain nucleic acids in this manner. Several of these nucleic acid crosslinking agents have been utilized in simple in vitro systems for the purposes of selectively crosslinking and inactivating a specific nucleotide sequence by means of affinity techniques. In such affinity inactivation procedures, the crosslinking agent is first attached through one of its functional moieties to a single-stranded oligonucleotide carrier chain. When this derivatized oligonucleotide carrier chain is contacted with a single-stranded oligonucleotide target chain having a nucleotide sequence complementary to that of the carrier chain, the target chain becomes base-paired to the carrier chain and then reacts with the free functional moiety of the crosslinking agent to thereby crosslink the two chains.
The nucleic acid crosslinking agents which have previously been proposed for use in affinity inactivation procedures, all have certain limitations which restrict their application to only certain types of nucleic acids and in only very simple in vitro systems. The major difficulty with all of these crosslinking agents lies in the particular reactive sites which they require on the polynucleotide carrier chain for successful attachment thereto. These particular attachment sites are either totally lacking in a vast number of nucleic acids or, if present, are such that they enable only one molecule of crosslinking agent to be attached per polynucleotide chain. This limitation precludes their use in complex in vitro systems or in vivo wherein it is desired to irreversibly inactivate a relatively long nucleotide sequence, since a single interstrand crosslink in such cases would not be likely to be sufficiently stable to effect irreversible inactivation due to depurination reactions and the ability of cells to repair low levels of interstrand crosslinks.
In attempting to overcome the above-described limitations and develop nucleic acid crosslinking agents having greater versatility in complex systems than those previously proposed, a number of important factors must be taken into consideration involving the proper selection, coordination and spacing of the attaching moiety (i.e., the functional group of the crosslinking agent which links to the attachment site of the polynucleotide carrier chain) and the crosslinking moiety (i.e., the functional group of the crosslinking agent which reacts at the crosslinking site of the polynucleotide target chain). First of all, the attaching moiety must be such that it will be reactive with one or more residues or groups common to a vast majority of nucleic acids and generally present therein in sufficient number so as to enable multiple attachment sites on the carrier chain. Secondly, the crosslinking moiety must be such that it will be unreactive with the carrier chain during attachment of the crosslinking agent to the carrier chain and hence will not form intrastrand crosslinks, but will be reactive with the target chain upon base-pairing of the two chains so as to form interstrand crosslinks. Thirdly, the attaching moiety and the crosslinking moiety must be properly coordinated and spaced with respect to each other that upon base-pairing of the derivatized carrier chain to the target chain, the crosslinking moiety of each attached molecule of crosslinking agent will be properly positioned with respect to a reactive crosslinking site on the target chain so that it may react therewith. In addition to these basic requirements, the crosslinking moiety should have sufficient stability and reaction specificity in biological systems so as to be capable of withstanding premature deactivation before the derivatized carrier chain has been able to become base-paired with its target chain. None of the previously proposed nucleic acid crosslinking agents has been able to satisfy all of the foregoing requirements.